Nunc immuno maxisorp elisa plate

Maintaining mucin homeostasis is vital for respiratory health (Ridley and Thornton 2018 (link)). The effects of CS on the intracellular expression and secretion of 3 airway mucin proteins, i.e., MUC5AC, MUC5B, and club cell secretory protein (CCSP) were examined at T12 and PT20 using an ELISA method. Apical washes were collected into 200 µL DPBS. The disulfide bonds in the mucins were disrupted by adding DTT to a final concentration of 0.025 mM. Cell debris was removed by centrifugation at 600 × g for 10 min at 4 °C. Mucin samples (50 µL apical washes for mucin secretion analysis and 3 µg whole cell lysates for mucin expression analysis) were loaded onto a NUNC-Immuno^® MaxiSorp™ ELISA plate (Thermo Fisher Scientific); the plate was dried in a 37 °C incubator overnight. Detection of the mucin proteins was conducted as described previously (Xiong et al. 2018 (link)).

Secretion and expression of MUC5AC, MUC5B, and club-cell secretory protein (CCSP) were measured in apical washes and cell lysates, respectively, using an ELISA method as described previously [34 (link)]. Apical washes (50 µL) or protein lysates (5 µg) were coated onto a NUNC-Immuno^® MaxiSorp™ ELISA plate (Thermo Fisher Scientific) at 37 °C overnight. Mucin proteins were detected by incubating for 90 min at room temperature with antibodies against MUC5AC (Pierce), MUC5B (Abcam), or CCSP (Millipore, Burlington, MA, USA), all of which were diluted at 1:500 in DPBS containing 10 mg/mL bovine serum albumin (BSA), 0.3% Triton X-100, and 0.2% Tween-20, followed by a 60-min incubation with HRP-conjugated goat-anti-mouse antibody (Rockland, Limerick, PA, USA) diluted at 1:1000 in the same buffer. Plates were washed three times with DPBS. Color was developed by adding 50 µL 3′,5′-tetramethylbenzidine (TMB, Thermo Fisher Scientific, USA) to each well and the reactions terminated with 50 µL Stop Solution (Abcam). Absorbance at 450 nm was measured using a Synergy H4 microplate reader.